1. Field of the Invention
The present invention relates to biomarker specific moieties conjugated quantum dots capable of highly sensitivity and selective biomarker detection.
2. Brief Description of the Prior Art
Breast cancer surgery relies on accurately assessing whether cancer cells are present in excised tissue to ensure complete tumor removal. As expected, the local cancer recurrence rate is higher for tissue margins testing positive for cancer cells within 3 mm of the margin surface (16%) in comparison to tissue margins that test negative (6%). Generally, re-excision is required if the tissue margin is found to contain cancer cells, which often results in additional cost, time, and pain to the patients. Estimates of the re-excision rate vary from 12% to as high as 60%. Morbidity and cancer recurrence rates were found to be much higher in patients who were re-excised. Consequently, it is highly desirable to cleanly remove a tumor with margins that test negative in a single surgical procedure.
Currently there is no reliable intraoperative margin assessment method. Conventional surgical procedures, such as preparation cytology (hereinafter “TPC”) and frozen section analysis (hereinafter “FSA”), do not provide sufficiently reliable findings. TPC assumes that tumor cells will adhere to a clean glass surface, while other cells will not. The slides are then stained and reviewed by cytopathologists. FSA involves taking 1 cm×1 cm tissue samples from the walls of a surgical cavity and staining these samples with hematoxyline and eosin. The reliability of FSA and TPC are questionable due to histological artifacts. Currently, only a pathology exam is able to accurately assess the tissue margin.
VEGF antigen has little expression in normal cells, with the exception of wound sites that require the formation of additional blood vessels. Instead, VEGF is primarily expressed in cancerous cells. For example, VEGF is usually observed in breast cancer tissue, although the expression varies depending upon the histological grade of the tumor cells. The distribution of VEGF positive cells is not uniform. The strong expression is concentrated mainly along the edges of invasive tumors. Consequently, it is particularly well suited for detecting the presence of cancer cells in the margin of excised tissue. VEGF expression is correlated to the degree of differentiation. For invasive ductal carcinomas (IDC), VEGF is expressed in about 40% of cancer cells at stage I, about 75.6% of cancer cells at stage II and about 77.8% of cancer cells at stage III. As the cancer cells become more differentiated, e.g. mature, VEGF expression increases. For the non-invasive type of breast cancer, ductal carcinomas in situ (DCIS), VEGF is expressed in about 93% to about 100% of cancer cells.
The structure of TACAs, such as Tn antigen, are derived during neoplastic transformation changes within the glycosylation pathways. Tn antigens are only expression in cancer cells and are homogenously expressed throughout a tumor. There is no expression of Tn antigen in normal breast tissues. Furthermore anti-Tn antigen can be used to distinguish between benign and malignant cancer cells. Between about 60% to about 80% cancer cells express Tn antigen in DCIS and about 20% to about 50% of cancer cells express Tn antigen in lobular carcinoma in situ. Expression in IDC varies according tissue differentiation; at stage I it is expressed in about 70% cancer cells, at stage II it is expressed in about 90% to about 100% of cancer cells, and at stage III it is expressed in about 40% to about 60% of cancer cells. In contrast to VEGF antigens, which are only weakly expressed in invasive lobular carcinoma, Tn antigens are expressed in about 20% to about 70% of cancer cells in invasive lobular carcinoma.
Recently, quantum dots (hereinafter “QDs”) have been proposed for use in various biomarker detection applications. WO/2008/140624, for example, discloses bioconjugated bifunctional nanoparticles for use in biomedical imaging applications. In some embodiments, nanoparticles are synthesized by a process that involves replacing a capping ligand such as mercaptopropionic acid (MPA) with another capping agent such as 3-mercaptopropyltrimethoxysilane (MPS). The nanoparticles may absorb light in the near-infrared optical region. To detect cancer cells, the nanoparticle may be conjugated with target moieties specific for a cancer biomarker, such as VEGF and EGFR. It is further contemplated that the nanoparticles may be used for intraoperative margin evaluation during surgery.
Danussi, Carla et. al. “A newly generated functional antibody identifies Tn antigen as a novel determinant in the cancer cell-lymphatic endothelium interaction,” Glycobiology, 19(10), pps. 1056-1067, October 2009 discloses a study using anti-Tn IgM antibody conjugated QDs to determine the presence of metastatic cells in vivo and by analyzing excised tissue. The article teaches that Tn is expressed by 90% of human carcinomas and Tn antigen is proposed as a diagnostic and prognostic tumor biomarker for breast carcinoma.
Other references teach the synthesis of metal sulfide quantum dots for use in bioconjugation. For example, U.S. Patent Application No. 2009/0286257 discloses aqueous QDs for use in bioconjugation, such as CdPbS QDs, that emit light in the near infrared region, and U.S. Patent Application No. 2009/0065742 discloses aqueous QDs, such as MPS-replaced CdS and MPS-replaced ZnS QDs for use in bioconjugation.
There is a need to develop an improved biomarker specific conjugated QD that is reliable, accurate and capable of highly sensitive and selective biomarker detection as well as an improved method for quantum dot conjugation. Furthermore, there is a need to develop improved conjugated QDs capable of quickly and reliably assessing the margin of excised tissues in real time, allowing a surgeon to perform tissue excision and re-excision in a single surgical procedure, thereby reducing patent morbidity, treatment delays, inconvenience, expense and pain.